Blood is made up of red blood cells (erythrocytes) carried in a solution called "plasma." Blood plasma includes numerous proteins which are useful for treating humans for various medical indications. Two examples of such proteins are fibronectin, also known as cold-insoluble globulin (CIg) and anti-hemophilic factor (AHF or Factor VIII).
Fibronectin is a multi-functional glycoprotein present in plasma at a concentration of approximately 300 micrograms per milliliter. Some of its biological properties include adhesion of cells to surfaces and binding to collagen, fibrin, and heparin. Reduced levels of fibronectin have been found in patients with trauma, burn injury, sepsis and severe malnutrition. Replenishment of fibronectin levels has been correlated with clinical improvement in such patients.
Fibronectin has been found to have a molecular weight of 440,000 daltons and to consist of two nearly identical 220,000-dalton subunit polypeptides. Fibronectin compositions or concentrates can be made up of varying ratios of both the dimer (the 440,000-Dalton unit) and the monomer (the 220,000-dalton unit). It has been suggested that the activity of a fibronectin composition is relatively higher when the proportion of dimer to monomer is relatively higher.
Factor VIII, which is associated with the coagulation or clotting of blood, is present at deficient levels in certain individuals, or is absent. For example, persons who have a deficiency (or absence) of antihemophilic factor (AHF or Factor VIII), i.e., persons suffering from hemophilia A, have blood which either fails to clot or clots only after longer periods of time than the time required for clotting in a person who has a normal level of Factor VIII.
In the past, persons suffering from hemophilia A (hemophiliacs) were treated by transfusing them with whole blood or blood plasma. More recently, however, Factor VIII concentrates have become available for administration to such persons. Such Factor VIII concentrates are produced by fractionating blood plasma into various components and recovering the components separately, including the Factor VIII component. Some processes for producing Factor VIII concentrate have been based on a discovery by Poole et al (Nature, Vol. 203, p. 312, 1964) that the precipitate remaining after plasma is frozen and then thawed, i.e., the cryoprecipitate, contains Factor VIII in a concentrated form and excludes various other protein fractions. It was discovered that, in addition to Factor VIII, the cryoprecipitate also includes the major portion of the fibronectin component of plasma.
Work progressed over the years to perfect the separation of Factor VIII from other proteins in the cryoprecipitate, including the fibronectin component, so that the resulting products would incorporate increased concentrations of Factor VIII relative to the other proteins present in the plasma.
One area in which a substantial amount of work has been done in production of Factor VIII is based on the use of polyethylene glycol (PEG) to precipitate Factor VIII from other proteins in an aqueous cryoprecipitate solution. For example, U.S. Pat. No. 3,652,530, which issued on Mar. 28, 1972 to A. J. Johnson, discloses a process for preparing a Factor VIII concentrate by fractionating cryoprecipitate with PEG at a relatively lower concentration to precipitate fibrinogen and other proteins, and then increasing the PEG concentration to precipitate Factor VIII. There is no disclosure of the fate of the fibronectin.
U.S. Pat. No. Re. 29,698 to Fekete et al discloses a process for production of Factor VIII (AHF) by which heparin is added to a cryoprecipitate solution, along with PEG, to provide increased yields of precipitated Factor VIII. It is disclosed that the amount of heparin employed during the fractionation step can vary, with the optimum concentration being 1 unit of heparin per ml of the plasma solution, whereas concentrations of heparin greater than about 10 units per ml are to be avoided as dangerous.
It is desired to provide a process for producing fibronectin and Factor VIII which results in increased purity, higher concentration, and enhanced yields of both proteins. Further, it is desired that the process provide a fibronectin concentrate comprising a maximized ratio of fibronectin dimer to monomer.